Nitric oxide has just recently been identified as a molecule which plays a fundamental role in biological processes. As a result, research into the physiology and pathology of nitric oxide has grown explosively. This research activity has, in turn, created a demand for accurate and precise techniques for the determination of nitric oxide (NO.), a free radical gas that is short-lived in biological materials.
Several methods for detecting nitric oxide in biology and medicine are well established. These include spectrophotometry, chemiluminescence, and paramagnetic resonance. These are ex situ techniques, however. That is, a sample of biological fluid, for example the extracellular fluid in a tissue or the support buffer in a suspension of cells, must be analyzed out of its biological context. The measurements made on such samples reflect nitric oxide concentration at a single time, and when assembled in a series make a discontinuous record. Therefore, these methods, though valuable, are not ideal for following rapid processes, because changes in nitric oxide concentration are not observed if they occur between sampling points. However, the ability to follow rapid changes is important because nitric oxide is unstable in the presence of oxygen, persisting but a few minutes or seconds in biological systems.
Recently, electrodes for the direct electrochemical detection of nitric oxide have been developed. The earliest of these electrodes, known colloquially as the "Shibuki electrode", is a modification of an electrode for detecting O.sub.2 and functions to exclude interfering species by means of a membrane permeable only to gases. See, K. Shibuki, Neuroscience Research, 9 (1990) 69-76 (the entire content of which is expressly incorporated hereinto by reference). The Shibuki electrode uses a Pt electrode to oxidize nitric oxide at 800 mV and to register the resulting oxidation current. This sensor is reported to have limited biological usefulness, because it does not respond linearly to nitric oxide concentrations greater than 1 .mu.M and is subject to a destructive buildup of the oxidation products of nitric oxide within the enclosed electrolyte surrounding the Pt electrode.
More recently, another method became available, using a metalloporphyrin membrane electrochemically deposited on a carbon fiber electrode. See, T. Malinski et al, Nature, vol. 358, 676-677 (1992); T. Malinski et al, "Nitric Oxide Measurement by Electrochemical Methods", Methods in Nitric Oxide Research, chapter 22 (1996); and Published International Patent Application No. WO 93/21518 to T. Malinski (the entire content of each publication being expressly incorporated hereinto by reference). This sensor is constructed by electrochemically depositing a metalloporphyrin, for example nickel-tetrakis (3-methoxy-4hydroxyphenyl) porphyrin, on a carbon electrode (which may be as small as a single carbon fiber a few .mu.m in diameter, or less). The porphyrin surface is then coated with a final layer of Nafion.TM. (Dupont), a fluorocarbon polymer that forms a network of interconnected cavities lined with sulfonate groups (SO.sub.3.sup.-). Cations and neutral solutes are conducted through the cavities with the anions being excluded. Direct measurements of nitric oxide have been reported using this porphyrinized electrode, with good sensitivity and selectivity. Furthermore, this porphyrinized electrode can be produced in micron or submicron tip diameters, suitable for extra- and intra-cellular measurements. Its disadvantages include the difficulty of handling micron-diameter carbon fibers, which require manipulation under a microscope with cold illumination (or under water) to eliminate thermal convection currents that disturb the fibers. Furthermore, the fibers, though strong for their size, are easily broken and are not degraded or absorbed in biological tissue. There are additional concerns about the exact chemical mechanism by which this electrode detects nitric oxide, since carbon fibers without a porphyrin coating or with a coating of porphyrin without a metal ligand can also detect nitric oxide with significant sensitivity.
A biochemically-modified electrode has also been proposed that employs Cytochrome c as a nitric oxide sensor, catalyzing the electrochemical reduction of nitric oxide and NO.sub.2.sup.- at -580 mV. See, K. Miki et al, Journal of Electroanalytical Chemistry, 6, 703-705 (1993) (the entire content of which is expressly incorporated hereinto by reference).
Nitric oxide-detecting electrodes have also been constructed with wires of precious metals, notably platinum and gold. See, F. Pariente et al, "Chemically modified electrode for the selective and sensitive determination of nitric oxide (NO) in vitro and in biological systems", Journal of Electroanalytical Chemistry, 379, 191-197 (1994) and F. Bedioui et al, "The use of gold electrodes in the electrochemical detection of nitric oxide in aqueous solution", Journal of Electroanalytical Chemistry, 377, 295-298 (1994) (the entire content of each publication being hereby expressly incorporated hereinto by reference). Furthermore, a porphyrinic-based platinum-iridium electrode has also been constructed and is available commercially. See, K. Ichimori et al, "Practical nitric oxide measurement employing a nitric oxide-selective electrode", Ref. Sci. Instrum., 65 (8) August 1994 and H. Miyoshi, FEBS Letters, 345, 47-49 (1994) (the entire content of each publication being hereby expressly incorporated hereinto by reference).
Thus, although there have been prior proposals in the literature, the development of electrodes for the electrochemical detection of nitric oxide has yet to reach the level that would allow widespread use in biomedical research. Such an electrode must be: (1) highly sensitive (with limits of detection for nitric oxide in the nanomolar range and below); (2) highly selective for nitric oxide against interfering anions; (3) easy to prepare reproducibly in very small diameters (10 .mu.m or less); (4) able to respond rapidly to nitric oxide, whose half-life is but a few seconds in physiological conditions; and (5) stable for minutes to hours in biological fluids and tissues. It is towards fulfilling such needs that the present invention is directed.